PROJECT SUMMARY Enhancing remyelination is a critical strategy for restoring brain function after demyelination in multiple sclerosis (MS) patients; however, despite concerted efforts, the ability to stimulate remyelination in MS brain has remained elusive. While signaling pathways that promote oligodendrocyte precursor differentiation have been identified, the experimental milieux under investigation do not replicate the mechanisms limiting remyelination following MS-specific inflammatory CNS injuries. The current proposal builds on our new models of demyelination/remyelination using pathogenic recombinant antibodies (rAbs) generated from MS patients. Myelin-specific MS rAbs direct complement-mediated demyelination in vivo and ex vivo, all of which spontaneously repair in association with microglial activation. However, demyelinated explants that are continuously exposed to myelin-specific MS rAb fail to activate microglia, and oligodendrocyte maturation is inhibited. Similarly, targeted depletion of microglia following rAb-mediated demyelination blocks oligodendrocyte maturation preventing active remyelination. Using single cell RNASeq (scRNASeq) on microglia isolated from remyelinating explants, we identified transcriptionally distinct microglial subsets that are associated with successful or failed remyelination. Hence, we hypothesize that microglial signals are critical for oligodendrocyte responses during the transition from early myelinating to actively myelinating oligodendrocyte, and myelin-specific MS autoantibody modulates these signals to arrest remyelination. To test our hypothesis, we propose three complementary specific aims. In Aim 1, we will evaluate microglial and oligodendrocyte responses in in vivo models of MS rAb-mediated demyelination and compare those responses to those seen in toxin-mediated demyelination. Intrathalamic or corpus callosum injection of myelin-specific MS rAb plus HC will be performed in conjunction with pharmacologic microglial depletion and chronic administration of MS rAb to validate the impact of microglial responses on remyelination in the intact nervous system. Comparable studies will be done following lysolecithin-induced demyelination, which has a very different time course of microglial activation and remyelination. In Aim 2, we will study the dynamics of demyelination, microglial responses and oligodendrocyte regeneration in situ using intravital imaging following cortical demyelination. This real-time analysis of myelin loss, microglial activation and remyelination will be compared to that seen following cuprizone-mediated demyelination. Finally, in Aim 3, we will investigate the mechanisms by which microglia impact remyelination using ex vivo cerebellar slices demyelinated with myelin- specific rAb plus human complement (HC). We will focus on investigating the role of several microglial genes identified by scRNASeq that are expected to promote or impair remyelination. Normal appearing white matter and MS lesion tissue with varying degrees of demyelination and remyelination will be evaluated to determine the abundance and localization of functionally-important microglial subsets. The results of these studies will provide insights into novel mechanisms controlling remyelination after inflammatory injury. In addition, the knowledge gained may identify novel therapeutic approaches that will result in clinically-meaningful myelin repair.